The Development Of RP-HPLC Method In Analysis Of Related Substances Of Small Molecule Drugs

In the process of drug research and development,quality standard,acting as the end-point control together with manufacture processing control,provides a strong assurance in the safety,effectiveness,and quality controllability.Related substances refer to organic impurities similar to or originally related to the main components of drugs.They are the important items in quality standards.It is one of key characteristics of every drug and also the focus and difficult point of quality research.The related substances are mostly determined by RP-HPLC method.This study focuses on the development of RP-HPLC method in analysis of related substances of small molecule drugs.Up to present,a common problem in related substances method development in domestic companies is to copy the existing methods recorded in the standards documents without considering whether the standards are applicable to their own products.Sometimes,just a simple parameter adjustment of reference or existing standard methods is made but no systematical validation performed so that some new impurities cannot be separated efficiently,as a result,a thorough modification of the method or even development of a new method from scratch are needed.Failure of method transfer due to lack of evaluation of the reproducibility,abandon of some inseparable impurities instead of updating the standardized methods under the conserved filing strategy carried out by some companies,are contrary to the original purpose of quality research itself.Chromatography is an experimental science and what we need is to solve the application problems under the existing conditions.To achieve this goal,this study is to develop an operational procedure for related substances determination in small molecule drugs using RP-HPLC technology under the current domestic laboratory condition,based on summarization of theoretical and practical operation,with aims at separation and reproducibility as the goal of method development from the application point of view,and comprehensive investigation of chromatographic characteristics of impurities,etc.To the problems currently in the related substances method development,four points of view are presented here: difference is the breakthrough of separation;separation is only the beginning;method is developed rather than validated;ideal method has good reproducibility.These ideas run through the development process of the method described in this paper.In the first chapter,the concept and importance of related substances are presented and the status and challenges in the development of related substance determination methods in China discussed,while four viewpoints that runs throughout method development are put forward and the content and the significance of this study concluded.In the second chapter,the application of chromatographic separation theory in practice is discussed with many studied cases and notes.Based on the theory and practice,this chapter primarily discusses the three most influential chromatographic factors: pH value of mobile phase,chromatographic column,and mobile phase composition.Secondly,gradient,column temperature and salt concentration are illustrated.It is emphasized that in the process of separation research,the first step is to find suitable separation conditions,and the next,to collect enough information,focusing on those impurities with different responses and their sensitive condition range,and to master the chromatographic separation characteristics of each impurity.Chapter 3 is about the systematic optimization of method based on reproducibility.Many of the methods developed by R&D laboratories need to be transferred to other collaborative laboratories,QC laboratories in manufacture,and even the labs in NIFDC,and will continue to be used throughout the drug life cycle.Therefore,how to ensure the reproducibility of methods in different laboratories at different times is a very important issue.This chapter summarizes the ways to improve the reproducibility of the method from many aspects with applicable procedures.At this stage,every potential factor that affects the reproducibility should be investigated and its fault tolerance improved which therefor,is a delicate optimization process.At the end of this chapter,the author innovatively gives a systematic development list in related substances determination methods for reference in practical work.Chapter 4 is a study of the points described in Chapter II,which elaborates the development process of impurity separation for a respiratory API.Firstly,the key chromatographic parameters were determined through the selection of mobile phase pH value and chromatographic column;secondly,the optimization of mobile phase composition and gradient was investigated to solve the problem of baseline interference;thirdly,column temperature and diluent were optimized;fourthly,the method was confirmed by destructive test;and finally,the ratio conversion of mobile phase was carried out to solve the problem that mobile phase could not be used for a long time,and in the same time the baseline was optimized.The whole process proves work well by comparing the self-developed method with the existing methods in pharmacopoeia.It turns out that the self-developed method is superior to the Pharmacopoeia method.Chapter 5 is a complete study described in Chapter 2 and Chapter 3.With the help of the method development list mentioned in Chapter 3,a comprehensive study on the separation and reproducibility of related substances in a depressurized API was carried out,and the chromatographic characteristics of impurities in the whole research process were summarized with a list.In the whole research process,the mobile phase pH and chromatographic column were screened,the composition and gradient of mobile phase were optimized,the column temperature was screened,and the method was preliminarily confirmed by destructive test;then the wavelength was confirmed,the mobile phase and pH value were simplified,the preparation method of sample was optimized,the solvent effect and stability of diluent were investigated,the batch difference of chromatographic column and instrument were also examined.The differences between them were assessed,and many other details affecting the reproducibility were investigated.Finally,the preliminary verification was completed,and the related substance determination method of the product was established.The complete development process described in this paper is applicable to all stages of API and pharmaceutical technology research,you can choose partial development or systematic and complete method development to meet your needs.Identifying the key points in the method development,making most of method development list,and summarizing the impurities chromatographic characteristics will have your work done in high efficiency.